One of the major research efforts in Cardiology has been to salvage myocardium at risk for necrosis in patients with acute myocardial infarction. While modern clinical treatment is aimed at establishing early coronary reperfusion, it is known that reperfusion itself can result in further myocardial damage. Free radical generation has been proposed as the central mechanism responsible for this reperfusion damage. Until recently, there was only indirect evidence for free radical generation in the heart based on beneficial effects of free radical scavengers. There was a great need for a direct technique of measuring free radical generation in the postischemic heart. Over the past 4 years since original funding of this grant, we have developed and applied EPR techniques to measure, quantitate, and characterize free radical generation in the isolated crystalloid perfused heart. We also developed new instrumentation enabling in-vivo EPR spectroscopy of whole beating hearts at L-band (1-2 GHz). The presence and time course of free radical generation in the isolated heart has been determined, however, little is known regarding the mechanisms which control this process and the effects of other in-vivo factors. In this renewal application we propose to continue our research to obtain a better understanding of the nature and mechanisms of postischemic free radical generation in the isolated heart, and to extend these observations in order to gain an understanding of the nature of free radical mediated reperfusion injury in-vivo. Direct, and spin trapping at X-band, as well as in-vivo EPR studies at L-band will be performed to measure, quantitate, and characterize free radical generation in the in- vivo rabbit heart and in isolated perfused hearts containing blood components. Free radical generation will be correlated with the observed reduction in cardiac contractile function, levels of high energy phosphates, and the metabolic state of the heart. These experiments should provide new fundamental insight into the nature and mechanisms of free radical generation in the postischemic heart.